5-Methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, in genetic imprinting and in tumorigenesis (or review see Millar et al.: Five not four: History and significance of the fifth base: in S. Beck and A. Olek, eds.: The Epigenome; Wiley-VCH Verlag Weinheim 2003, S. 3-20). The identification of 5-methylcytosine as a component of genetic information is thus of considerable interest. 5-Methylcytosine positions, however, cannot be identified by sequencing, because 5-methylcytosine has the same base-pairing behavior as cytosine. Additionally, in the case of a PCR amplification, the epigenetic information borne by 5-methylcytosines is completely lost.
The usual methods for methylation analysis operate essentially according to two different principles. Either methylation-specific restriction enzymes are utilized, or a selective chemical conversion of unmethylated cytosines to uracil is conducted (bisulfite treatment). The enzymatically or chemically pretreated DNA is then amplified and can be analyzed in different ways (or review see Fraga and Esteller: DNA Methylation: A Profile of Methods and Applications:Biotechniques 33:632-649, September 2002; and see also WO 02/072880 pp. 1 ff).
As the use of methylation-specific enzymes is restricted to certain sequences containing restriction sites recognized by said enzymes, for most applications a bisulfite treatment is conducted for review see U.S. patent application Ser. No. 10/311,661).
According to the invention described herein below, a “bisulfite reaction”, “bisulfite treatment” or “bisulfite method” refers to a reaction for the conversion of cytosine bases in a nucleic acid to uracil bases in the presence of bisulfite ions, whereby 5-methyl-cytosine bases are not significantly converted. The bisulfite reaction contains a deamination step and a desulfonation step which can be conducted separately or simultaneously (further details are described, and a reaction scheme is shown in EP 1394172A1, incorporated by reference herein in its entirety). There are various documents addressing specific aspects of the bisulfite reaction, including Hayatsu et al., Biochemistry 9:2858-28659, 1970; Slae and Shapiro, J. Org. Chem. 43:4197-4200, 1978; Paulin et al., Nucl. Acids Res. 26:5009-5010, 1998; Raizis et al., Anal Biochem. 226:161-1666, 1995; and Wang et al. Nucleic Acids Res. 8:4777-4790, 1980, and these documents, summarized in EP 1394172A1 are also incorporated by reference herein in their entirety.
The bisulfite treatment is usually conducted in the following way: The genomic DNA is isolated, mechanically or enzymatically fragmented, denaturated by NaOH, converted several hours by a concentrated bisulfite solution and finally desulfonated and desalted (e.g.: Frommer et al.: A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA. 89:1827-31, 1992, incorporated by reference herein in its entirety).
In recent times several technical improvements of the bisulfite methods were developed. The agarose bead method incorporates the DNA to be investigated in an agarose matrix, through which diffusion and renaturation of the DNA is prevented (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps are replaced by rapid dialysis (Olek A. et al. A modified and improved method for bisulphite based cytosine methylation analysis, Nucl. Acids Res. 24:5064-5066, 1996). In the patent application WO 01/98528 (=DE 100 29 915; =U.S. application Ser. No. 10/311,661), a bisulfite conversion is described in which the DNA sample is incubated with a bisulfite solution of a concentration range between 0.1 mol/1 to 6 mol/1 in presence of a denaturing reagent and/or solvent and at least one scavenger. In said patent application several suitable denaturing reagents and scavengers are described (document incorporated by reference herein in its entirety). In the patent application WO 03/038121 (=DE 101 54 317; =Ser. No. 10/416,624) a method is disclosed in which the DNA to be analysed is bound to a solid surface during the bisulfite treatment. Consequently, purification and washing steps are facilitated. Further improvement are described in the patent applications EP1394173A1 and EP1394172A1 (incorporated by reference herein in its entirety).
However, a basic problem of the bisulfite treatment consists of the fact that long reaction times are necessary in order to assure a complete conversion and to exclude false-positive results. Simultaneously, however, this leads to a degradation of the DNA due to the long reaction times. Higher reaction temperatures in fact lead to a higher conversion rate, but also to a more intense degradation of the DNA. The interactions between temperature, reaction time, rates of conversion and degradation were recently investigated systematically. In this way, it could be shown that the highest conversion rates were attained at temperatures of 55° C. (with reaction times between 4 and 18 hours) and at 95° C. (with a reaction time of one hour). A serious problem, however, is the degradation of the DNA during this procedure. At a reaction temperature of 55° C., 84-96% of DNA is decomposed. At 95° C. the degradation is in fact even higher (Grunau et al.: Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 29:E65-5, 2001; incorporated by reference herein in its entirety). Thus, most authors use reaction temperatures of approximately 50° C. (see Frommer et al., loc. cit. 1992, p. 1827; Olek et al., loc. cit. 1996, p. 5065; Raizis et al: A bisulfite method of 5-methylcytosine mapping that minimizes template degradation, Anal Biochem 226:161-6, 162, 1995).
In addition to the high degradation rate of DNA, there is another problem in conventional bisulfite methods, which consists of the fact that a powerful purification method for converted DNA has not yet been described. Many authors use precipitations (see Grunau et al., oc. cit). A purification via DNA-binding surfaces has also been described (see Kawakami et al.: Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma, Journal of the National Cancer Institute, 92:1805-11, 2000). The yield of these purifications, however, is limited.
Due to the high losses of the conventional bisulfite treatment, it is problematic to use these methods for investigations in which the quantity of DNA to be analyzed is limited. A particularly interesting field of methylation analysis, however, lies in diagnosing cancer diseases or other disorders associated with a change in methylation status by means of analysis of DNA from bodily fluids, e.g. from blood or urine. However, DNA is present only in small concentrations in body fluids, so that the applicability of methylation analysis is limited by the low yield of conventional bisulfite treatment.
Accordingly, based on the particular importance of cytosine methylation analysis and based on the described disadvantages of conventional methodology, there is a great technical need for improved methods of bisulfite conversion.